1. Field of the Invention
The present invention is directed to methods and compositions for prophylaxis, treatment and diagnosis of pneumonic pasteurellosis in cattle. More particularly, the present invention relates to the identification and isolation of Pasteurella haemolytica antigens, using both recombinant DNA and non-recombinant technology, and the use of such antigens in the formulation of antigen and antibody-containing compositions for the treatment and diagnosis of pasteurellosis.
2. Description of the Related Art
Pneumonic pasteurellosis, commonly referred to as Shipping Fever, is a major cause of economic loss in feedlot cattle. While there is evidence to suggest that several etiologic agents, for example, combinations of stress, respiratory viruses, and various bacteria may participate in this disease, Pasteurella haemolytica, serotype A1, appears to be the major cause of the severe fibrinous pneumonia which can be seen.
The pathogenesis of the disease is poorly understood. Overgrowth of the lung with P. haemolytica with resultant bronchial pneumonia is thought to be at least partly caused by a preceding vital infection. Studies have suggested that parainfluenza-3 virus can impair pulmonary clearance of P. haemolytica. Moreover, infectious bovine rhinotracheitis virus has been shown to predispose pulmonary infection with P. haemolytica. In experimental bacterial infections in mice which have been pre-infected with virus, it has been shown that protection can be afforded by prior inoculation with either the vital or bacterial agent.
However, attempts to protect cattle by immunization with respiratory vital vaccines and Pasteurella bacterins have generally proved unsuccessful. It has been proposed that antigenic challenge with dead bacteria, as is the case with bacterin immunization, is insufficient due to the nature of the P. haemolytica infection--live P. haemolytica apparently produce a cytotoxin having specificity for ruminant leukocytes. Thus, it is posited that following infection with P. haemolytica, the infected cow's immune system is suppressed to the extent that effective immunosurveillance is compromised and the infective organism can not effectively be challenged. The failure of Pasteurella bacterins to provide an effective immunization has been partly ascribed to the absence of sufficiently antigenic amounts of this leukotoxin in the bacterin preparation. The cytotoxin is thus believed to contribute to the pathogenesis of pneumonic pasteurellosis by impairing primary lung defense and subsequent immune response, or by induction of inflammation as a consequence of leukocyte lysis.
The physicochemical nature of the leukotoxin is only poorly understood. As noted, this toxin exerts no toxic effects on non-ruminant leukocytes. However, the toxic effects of the toxin on ruminant leukocytes is dose dependant--at lower doses, generally only subtle alterations in various metabolic processes are noted, whereas higher concentrations can result in loss of membrane integrity and cell death. Apparent species specificity of the leukotoxic effects of living P. haemolytica, and cell-free P. haemolytica supernatants, supports the hypothesis that the leukotoxin itself is involved in determining the species specificity of the Pasteurella-induced pneumonia. Moreover, experimental evidence from studies of the interactions of P. haemolytica and its culture supernatant with ruminant alveolar macrophages, peripheral blood monocytes, neutrophils, and lymphocytes suggests that P. haemolytica leukotoxin is important for successful colonization and growth of P. haemolytica in pulmonary tissues. Thus, cytotoxic effects of the leukotoxin for leukocytes in pulmonary tissues probably contribute to the pathogenesis of the disease.
In contrast with bacterin immunization, immunization protocols employing live P. haemolytica, and various protein extracts of P. haemolytica, have been shown to protect cattle against experimental challenge exposure to the bacterium. However, most of these studies involved experimental challenge exposure to live P. haemolytica organisms, in either mice, where the organism induces a septicemia rather than a respiratory syndrome, or cattle, where the organism is aritifically introduced into the cattle's lungs. As such, neither of these test systems represent a natural disease state, and are thus not believed to entirely correspond to natural pasteurellosis.
In 1985, Confer and Lessley investigated a series of saline protein capsular extracts of P. haemolytica and identified a number of antigen groups through immunoreaction with immune sera obtained from cows which had been immunized with live P. haemolytica organisms (Vet. Immunol. and Immunopath., vol. 10, pp. 265 and 279). Antibody response to immunization with various of these capsular extracts was found to correlate with resistance to an experimental challenge of P. haemolytica organisms. However, as noted, these studies involved the use of capsular (i.e.--cell membrane) proteins which were then immunoidentified using experimentally induced antisera rather than antisera from pasteurellosis-infected cattle. Moreover, it is believed that the use of capsular proteins, rather than secreted proteins, and the use of experimentally induced antisera, rather than antisera from diseased cattle, represent inherent drawbacks to such an approach to the identification of antigens useful in the treatment of the disease.
Thus, attempts to develop a pasteurellosis vaccine to date have centered on identifying the leukotoxin, or identifying antigens from protein extraction of the P. haemolytica cell itself, rather than identifying antigenic elements present in cell-free supernatants. However, the present invention, rather than focusing primarily on the leukotoxin, embodies the realization that cell-free supernatants contain numerous antigens--antigens which are necessarily absent or only minimally represented in P. haemolytica bacterins--which should serve to induce a more effective immunization, or serve to complement, and thereby improve, bacteria preparations. Moreover, the present invention embodies the further realization that effective P. haemolytica antigens should be identified using antisera obtained from naturally-infected, active or convalescent, cattle. The ultimate goal, therefore, is to achieve an antigenic composition which comprises a mixture or admixture of individual, relatively purified, P. haemolytica antigens which correspond, at least in terms of antigenic determinants, to antigens identified by antibodies present in naturally-infected antisera.
The present invention is thus directed in general to improved methods for identifying useful P. haemolytica antigens, one of which utilizes antisera from naturally-infected cattle to select antigens from cell-free P. haemolytica culture supernatants, and the other employing recombinant DNA technology to provide novel recombinant cells which are selected based on their ability to produce individual P. haemolytica antigens as identified by the antisera.